Supporting Information
Kim et al.
Aberrant ribosome biogenesis activates c-Myc and ASK1 pathways resulting
in p53-dependent G1 arrest
[SI Materials and Methods]
Reagents and antibodies
Antibodies were purchased from Santacruz, Cell Signaling, Sigma, Novus,
AdipoGen (Korea University) and Chemicon as described in Supplementary Table
S1. MG132, SB202190, SB203580, cycloheximide and RNasin were obtained from
Calbiochem (EMD Biosciences, La Jolla, CA). Lipofectamine™ and Lipofectamine™
RNAiMAX were purchased from Invitrogen. Protein A-Sepharose and ECL reagents
purchased from Roche Molecular Biochemicals (Mannheim, Germany).
Cell Culture
Human fibrosarcoma HT1080, normal human fibroblast F65 and immortalized
MEFs were maintained in DMEM (WelGENE Inc., Korea), and colon carcinoma
HCT116 cells were maintained in McCoy's 5a (WelGENE Inc.) supplemented with
10% fetal bovine serum (FBS, WelGENE Inc.) and 1% antibiotic solution containing
penicillin and streptomycin (WelGENE Inc.). Cells were grown at 37°C in a
humidified atmosphere of 5% CO2.
Immunoblotting and Immunoprecipitation
Cells were harvested and lysed on ice in TNN (20 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 50 mM NaF and 1 mM Na3VO4) or adequately constituted buffers containing
protease inhibitors (1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin and 1 µg/ml
pepstatin A). Cell extracts were quantitatively analyzed by Bradford assay (Bio-Rad).
Equal amounts of extract were resolved by 10~15% SDS-PAGE and subjected to
immunoblot analysis. For immunoprecipitation, we used about 1~2 μg of primary
antibody.
Immunofluorescence
HT1080 cells grown on cover slips were transfected with specific siRNAs. After 48
h, cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1%
Triton X-100 in PBS for 10 min at room temperature. Fixed cells were pretreated with
2% BSA in PBS for 1 h and incubated with specific antibodies for 1 h. Cells were
washed three times in PBS and stained with Alexa-488 (anti-rabbit) and Alexa-568
(anti-mouse)-conjugated goat antibodies (Invitrogen). The images were obtained
using a LSM510 META confocal microscope (Carl Zeiss MicroImaging).
Metabolic labeling
siRNA-transfected cells were starved with methionine-free DMEM for 1 h and then
pulse-labeled with 50 μCi/ml [35S] methionine for 30 min. Cells were rinsed twice with
PBS and incubated in complete media, followed by the harvesting of in a timedependent manner. Resolved metabolic-labeled proteins were dried and visualized
with a BAS2500 imaging analyzer (Fujifilm, Tokyo, Japan).
To determine the stability of p53 in siRNA-treated cells, p53 proteins were
immunoprecipitated with p53 antibody and [35S] methionine-labeled p53 was
detected by a BAS2500 imaging analyzer (Fujifilm, Tokyo, Japan).
RNA preparations and Northern blotting
Total RNA was isolated from siRNA-transfected HT1080 cells using TRIzol
reagent according to manufacturer’s recommendations (Invitrogen). Equal amounts
of total RNA (2 μg) were denatured by heating at 65°C and separated using a 1%
agarose-formaldehyde gel containing ethidium bromide.
Synthesis of the ITS-1 and ITS-2 oligonucleotide probes (Supplementary Table
S2) was performed by annealing with two complementary primers, followed by endlabeling with T4 polynucleotide kinase and 10 μCi of [ 32P]-ATP. RNAs were
electrophoresed using a 1% agarose–formaldehyde gel and transferred to Nylon
membranes overnight. Hybridization of the membrane with the ITS-1 and ITS-2
oligonucleotides was performed as previously described (Robledo et al., 2008). The
sequence locations of the probes are as follows, ITS-1; 5’-end of internal transcriber
spacer 1, ITS-2; 5’-end of internal transcriber spacer 2. The RNAs hybridized with
the oligonucleotide probes were analyzed using a phosphorimager.
Thymidine incorporation assay
HT1080 cells, transfected with control or rpS3 siRNA, were rinsed with PBS and
then added to media containing 0.5 μCi/ml [3H]-thymidine and incubated for 3 h,
followed by two more washings with ice cold PBS. To remove remnants of [ 3H]thymidine in the cytoplasm, cell were incubated with 5% TCA on ice for 30 min and
then washed twice with ice cold PBS. Cells were lysed with lysis buffer (0.2 N NaOH
and 0.5% SDS) and incorporation of [3H]-thymidine was determined by a liquid
scintillation counter (Beckman). Each experiment was performed four times.
FACS analysis
siRNA-transfected HT1080, HCT116, F65 or MEF cells were fixed with cold 70%
ethanol for 1 h at -20°C and then washed twice with ice cold PBS by centrifugation.
Cell pellets were resuspended in 0.5 ml of PBS containing 1 mg/ml RNase A and
incubated for 30 min at 37°C. Following cell staining with 50 μg/ml propidium iodide
(Sigma), the cell cycle distribution was analyzed using a FACSCalibur flow cytometer
(BD Bioscience) and analyzed with CELLQuest software (BD Bioscience).
siRNA and Transfection
We designed or purchased siRNA oligos as described in Supplementary Table S1.
Specific siRNAs were reversely transfected using Lipofectamine™ RNAiMAX
transfection reagent according to manufacturer’s recommendations (Invitrogen).
DNA transfection was also performed using Lipofectamine™ (Invitrogen).
Statistical Analysis
Data are presented as mean values ± s.e.m. for at least three separate
experiments. Statistically significant differences between data sets were determined
using paired Student's t test.
[SI Figures and Tables]
Figure S1. Regulation mechanism of p53 level in rpS3 knockdown cells.
(A) Control or rpS3 siRNA-transfected cells were fractionated into cytosol and
nuclear fractions, respectively. Tubulin and PARP antibodies were used as fraction
markers for the cytosolic and nuclear fractions. (B) Total RNA was obtained from
HT1080 cells transfected with siRNAs. These isolated RNAs were reversetranscribed and then amplified by PCR with p53, rpS27 and rpS3 specific primers,
respectively. (C) siRNA-treated cells were subjected to metabolic labeling with [ 35S]methionine followed by incubation for 30 min. Labeled cells were lysed and extracts
were immunoprecipitated using p53 antibody. Total cell extracts (upper) and p53
immunoprecipitates (lower) were resolved by SDS-PAGE while [35S] methionine-
labeled proteins were visualized using a phosphorimager. (D) HT1080 cells were
transfected with siRNAs against control or rpS3 and then treated with MG132 (20
μM) for 4 h. Cell extracts were resolved and immunoblotted with indicated antibodies.
(E) HT1080 cells transfected with control or rpS3 siRNA were pulse-labeled with [35S]
methionine for 30 min and chased at the indicated times. Labeled p53 proteins were
immunoprecipitated using p53 antibody and resolved by 10% SDS-PAGE.
Figure S2. Large ribosomal proteins in 40S RP-depleted cells remain
unchanged.
(A) HT1080 cells, transfected with control or rpS3 siRNAs, were lysed and the cell
extracts were loaded onto a sucrose cushion. Proteins were then precipitated by
ultracentrifugation as described in Experimental Procedures. Equal amounts of
protein from the lysates and ribosome pellet were detected by immunoblotting.
Tubulin was used as both a loading control and marker protein for the non-ribosomal
fraction. (B) To separate each fraction, cell lysates used in (A) were ultracentrifuged
using a 20% (w/v) sucrose cushion as described in Experimental Procedures. Lysate,
non-ribosomal soluble fraction (Non-R), middle fraction (Mid-R) and ribosomal
fractions (R-pellet) were resolved by 12% SDS-PAGE respectively.
Figure S3. p53 induction is abolished by p38 inhibitor.
HT1080 cells were transfected with the indicated siRNAs. After 24 h, cells were
additionally untreated or treated with 20 μM SB202190 for 24 h respectively. Cell
extracts were resolved and subjected to immunoblot analysis using the indicated
antibodies.
Figure S4. G1 arrest due to rpS3 knockdown is mediated by p53 induction.
HCT116 (p53+/+) and HCT116 (p53-/-) cells were transfected with control or rpS3
siRNA. Cell cycle distribution was analyzed by flow cytometry (left). The percentage
of cells in G1 phase is shown in a graph (right). Data are means ± SD of values from
three independent experiments. *, p < 0.005 compared with control siRNA in
HCT116 (p53+/+) cells.
Figure S5. Protein synthesis and translation level of p53 in 40S RP-depleted
cells.
(A) HT1080 cells were transfected with siRNA as indicated. To identifiy the neosynthesized protein, the cells were incubated with [35S] methionine for 30 min. [35S]labeled proteins were visualized or quantified using either a phosphorimager (left) or
a scintillation counter (right), respectively. Data are means ± SD of values from four
independent experiments (*, P < 0.0001). (B) HT1080 cells, transfected with
indicated siRNAs, were subjected to metabolic labeling with [35S]-methionine as
described. Cells were lysed and extracts were immunoprecipitated using p53
antibody. p53 immunoprecipitates were resolved, and then [35S]-labeled proteins
were visualized using a phosphorimager (upper). Immunoprecipitated p53 protein
was detected by immunoblot analysis using p53 antibody (lower).
Figure S6. c-Myc is involved in the p53 induction and is downregulated by
feedback inhibition.
(A) HT1080 cells were transfected with HA-tagged p53 plasmid, and followed by
immunoblot analysis using indicated antibodies. (B) Cell lysates of HT1080 cells
transfected with control, rpS3 or rpS6 siRNA in combination with either p38 or c-Myc
siRNA were immunoblotted with indicated antibodies.
Figure S7. p14ARF induction and ASK1 reduction in response to impaired
ribosome biogenesis.
(A) HT1080 cells were transfected with control, rpS3 or rpS6 siRNA and harvested
as indicated times. Equal amounts of cell lysates were immunoblotted with indicated
antibodies. (B) HT1080 cells were transfected with siRNAs against negative control,
rpS3, rpS6 or RACK1. After 48 h, cells were fixed and immunostained with p14 ARF
(green) and p53 (red) antibodies. (C) HT1080 cells transfected with siRNAs against
control, rpS3, rpS6 or RACK1 were incubated, and followed by immunoblot analysis
using indicated antibodies and ASK1 kinase assay using GST-MKK6 as a substrate.
Figure S8. The induction of Arf in the S6 knockdown cells.
HT1080 cells were transfected with control and rpS6 siRNAs and harvested as
indicated times. Equal amouts of cell lysates were immunoblotted with indicated
antibodies.
Figure S9. The association of rpL11 with mdm2 increased in the RP-depleted
cells as well as p14 ARF.
Each siRNA-treated cells were lysed and extracts were immunoprecipitated using
indicated antibodies. Total cell extracts (left) and these immunoprecipitates (right)
were resolved by SDS-PAGE.
Figure S10. The translational level of c-Myc decreases in the aberrant
ribosomal biogenesis.
HT1080 cells were transfected with siRNA as indicated. After 12h, these cells were
treated with MG132 (right panel; 20 μM, 3 h) and the equal amouts of cell lysates
were immunoblotted with indicated antibodies.
Figure S11. The cellular response to ribosomal stress in the mouse embryonic
fibroblasts.
(A) MEF cells were transfected with siRNAs against negative control, rpS6 or
RACK1. After 24 h, cells were treated with 20 μM SB202190 for 24 h. (B) MEF cells
were firstly transfected with control, p38 or ASK1 siRNA. After 24 h, cells were
transfected with control or rpS6 siRNA and additionally incubated for 30 h. Cell cycle
analysis was performed using flow cytometry. Data are means ± SD of values from
three independent experiments (*, P < 0.05).
Figure S12. Alteration of the ribosome biogenesis by actinomycin D also
activates p38 MAPK.
HT1080 cells were treated with actinomycin D as indicated for 24 h. Immunoblot
analysis and ASK1 immunoprecipitation were performed with equal amounts of cell
lysates. The arrow indicates phosphorylation of ASK1.
Figure S13. Verification of p53 levels in the siRNA-treated cells.
Each siRNA-treated cell was lysed and immunoblot analysis was performed with
equal amounts of cell lysates.
Table S1. Antibodies and siRNAs used in this study.
Table S2. PCR primers and northern probes used in this study.